RESUMO
Mutation of RPE65 can cause severe blindness from birth or early childhood, and RPE65 protein is associated with retinal pigment epithelium (RPE) vitamin A metabolism. Here, we show that Rpe65-deficient mice exhibit changes in retinal physiology and biochemistry. Outer segment discs of rod photoreceptors in Rpe65-/- mice are disorganized compared with those of Rpe65+/+ and Rpe65+/- mice. Rod function, as measured by electroretinography, is abolished in Rpe65-/- mice, although cone function remains. Rpe65-/- mice lack rhodopsin, but not opsin apoprotein. Furthermore, all-trans-retinyl esters over-accumulate in the RPE of Rpe65-/- mice, whereas 11-cis-retinyl esters are absent. Disruption of the RPE-based metabolism of all-trans-retinyl esters to 11-cis-retinal thus appears to underlie the Rpe65-/- phenotype, although cone pigment regeneration may be dependent on a separate pathway.
Assuntos
Proteínas do Olho/fisiologia , Proteínas , Retina/fisiologia , Visão Ocular/fisiologia , Vitamina A/biossíntese , Animais , Proteínas de Transporte , Ésteres , Proteínas do Olho/genética , Camundongos , Camundongos Knockout , Microscopia Eletrônica , Mutação , Fenótipo , Retina/ultraestrutura , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/fisiologia , Rodopsina/metabolismo , Vitamina A/metabolismo , cis-trans-IsomerasesRESUMO
The disposal phase of the retinal rod outer segment renewal process has been studied by radioautography in adult frogs injected with tritiated amino acids. Shortly after injection, newly formed radioactive protein is incorporated into disc membranes which are assembled at the base of the rod outer segment. During the following 2 months, these labeled discs are progressively displaced along the outer segment owing to the repeated formation of newer discs. When the labeled membranes reach the end of the outer segment, they are detached from it. They subsequently may be identified in inclusion bodies within the pigment epithelium by virtue of their content of radioactivity. These inclusions have been termed phagosomes. Disc membrane formation is a continuous process, but the detachment of groups of discs occurs intermittently. The detached outer segment fragments become deformed, compacted, undergo chemical changes, and are displaced within the pigment epithelium. Ultimately, the material contained in the phagosomes is eliminated from the cell. In this manner the pigment epithelium participates actively in the disposal phase of the rod outer segment renewal process.
Assuntos
Membrana Celular/metabolismo , Células Fotorreceptoras/citologia , Pigmentos da Retina/fisiologia , Animais , Anuros , Autorradiografia , Epitélio/fisiologia , Microscopia Eletrônica , Biossíntese de Proteínas , TrítioRESUMO
Visual cell outer segment renewal was studied in eyes of mutant Royal College of Surgeons (RCS) and Sprague-Dawley (control) rats by a combination of microscopy and radioautography with the light and electron microscopes. RCS and control rats were injected with amino acids-(3)H at 11 days of age. Radioactive rod outer segment discs were assembled at the outer segment base from radioactive proteins synthesized in the rod inner segments. In controls, all radioactive discs assembled at 11 days of age were displaced the length of the outer segments, removed from outer segment tips, and phagocytized by the pigment epithelium by 8 days after injection. In the RCS rats, disc assembly and displacement resembled controls for the first 3 days after injection. However, as disc assembly continued for some time thereafter, a layer of labeled, disorganized, lamellar debris accumulated between the outer segment tips and the pigment epithelium. The buildup of debris was accompanied by visual cell death. At no time during the study was there evidence for phagocytic activity by the pigment epithelium. 61 days after injection, the layer of debris was the only heavily radioactive component in the retina. In the retina of RCS rats, the outer segment renewal mechanism malfunctions because the pigment epithelium does not fulfill its normal phagocytic role. The end result is visual cell death and blindness.
Assuntos
Células Fotorreceptoras/citologia , Células Fotorreceptoras/patologia , Retina/citologia , Retina/patologia , Degeneração Retiniana/etiologia , Degeneração Retiniana/genética , Pigmentos da Retina , Fatores Etários , Aminoácidos/metabolismo , Animais , Autorradiografia , Núcleo Celular , Citoplasma , Células Epiteliais , Epitélio/patologia , Histocitoquímica , Métodos , Microscopia Eletrônica , Fagocitose , Biossíntese de Proteínas , Ratos , Regeneração , Retina/metabolismo , Fatores de Tempo , TrítioRESUMO
Isolated frog retinas were incubated in vitro with a 4-h pulse of [3H]leucine, then chased for 32 h with a nonradioactive amino acid mixture. At the end of the incubation, light and electron microscope autoradiograms were prepared from some of the retinas. The autoradiograms revealed: (a) intense radioactivity in the basal disks of the rod outer segments, (b) diffuse label evenly distributed throughout the rod outer segments, and (c) a high concentration of label in the entire rod outer segment plasma membrane. Incubation under identical conditions, but with puromycin added, significantly inhibited the labeling of all of these components. To identify the labeled proteins, purified outer segments from the remaining retinas were analyzed biochemically by SDS disc gel electrophoresis and gel filtration chromatography. SDS gel electrophoresis showed that about 90% of the total rod outer segment radioactivity chromatographed coincident with visual pigment, suggesting that the radiolabeled protein in the plasma membrane is visual pigment. Gel filtration chromatography demonstrated that the radiolabeled protein co-chromatographed with rhodopsin rather than opsin, and that the newly synthesized visual pigment is both the basal disks and the plasma membrane is present in the native configuration.
Assuntos
Células Fotorreceptoras/ultraestrutura , Pigmentos da Retina/análise , Rodopsina/análise , Animais , Autorradiografia , Membrana Celular/ultraestrutura , Núcleo Celular/ultraestrutura , Mitocôndrias/ultraestrutura , Células Fotorreceptoras/análise , Puromicina/farmacologiaRESUMO
The permeability properties of frog rod photoreceptor outer segment discs were investigated in preparations of purified, dark-adapted, outer segment fragments by the techniques of direct volume measurement and electron microscopy. Outer segment discs were found to swell and contract reversibly in response to changes in the osmotic pressure of the bathing medium in accordance with the Boyle-van't Hoff law. By use of the criterion of reversible osmotic swelling, the disc membrane is impermeable to Na(+), K(+), Mg(+2), Ca(+2), Cl(-), and (PO(4))(-3) ions, whereas it is freely permeable to ammonium acetate. The disc membrane is impermeable to sucrose, although its osmotic behavior towards this substance is different from its behavior towards impermeable ions. Electron microscopy showed that the osmotic effects on the rod outer segment fragments represent changes in the intradiscal volume. Fixation with glutaraldehyde did not abolish the permeability properties of the disc membrane, and fixed membranes were still capable of osmotic volume changes. It is concluded from this study that the frog's rod photoreceptor outer segment discs are free-floating membranous organelles with an inside space separate and distinct from the photoreceptor intracellular space.
Assuntos
Concentração Osmolar , Células Fotorreceptoras/citologia , Acetatos/farmacologia , Animais , Anuros , Cálcio/farmacologia , Membrana Celular , Permeabilidade da Membrana Celular , Centrifugação com Gradiente de Concentração , Cloretos/farmacologia , Histocitoquímica , Junções Intercelulares , Magnésio/farmacologia , Microscopia Eletrônica , Fosfatos/farmacologia , Células Fotorreceptoras/efeitos dos fármacos , Potássio/farmacologia , Proteínas/análise , Compostos de Amônio Quaternário/farmacologia , Sódio/farmacologia , Coloração e RotulagemRESUMO
The in situ distribution of the 26-kdalton Main Intrinsic Polypeptide (MIP or MP 26), a putative gap junction protein in ocular lens fibers, was defined at the electron microscope level using indirect immunoferritin labeling of ultrathin frozen sections of rat lens. MIP was found distributed throughout the plasma membrane of the lens fiber cell, with no apparent distinction between junctional and nonjunctional membrane. MIP was not detectable in the basal or lateral plasma membrane of the lens epithelial cell, including the interepithelial cell gap junctions; nor was MIP detectable in the plasma membrane or gap junctions of the hepatocyte. Previous reports have indicated that the protein composition of the lens fiber cell junction differs from that of the hepatocyte gap junction. The evidence presented here suggests that the composition of the fiber cell junction and plasma membrane is also immunocytochemically distinct from that of its progenitor, the lens epithelial cell.
Assuntos
Proteínas do Olho/análise , Cristalino/ultraestrutura , Glicoproteínas de Membrana , Animais , Aquaporinas , Bovinos , Ferritinas/imunologia , Histocitoquímica , Técnicas Imunológicas , Microscopia Eletrônica , RatosRESUMO
Plasma membranes of vertebrate lens fiber cells contain a major intrinsic polypeptide with an apparent molecular weight of 26,000 (MIP26). These plasma membranes are extremely rich in communicating junctions, and it has been suggested that MIP26 is a component of them. MIP26 was purified from cow lenses using preparative SDS gel electrophoresis followed by hydroxylapatite column chromatography. From gel electrophoresis patterns and aggregational properties it was concluded that the MIP26 preparation was homogeneous. The purified MIP26 was used to produce monospecific antibodies in rabbits as assessed by double immunodiffusion and crossed immunoelectrophoresis of purified MIP26 and solubilized lens plasma membranes against the antiserum. Indirect immunocytochemical studies were performed on open and closed lens plasma membrane vesicles by incubation in anti-MIP antiserum followed by ferritin-conjugated goat antirabbit IgG. The conjugate bound unequivocally to lens communicating junctions, indicating that MIP26 is a component of these structures.
Assuntos
Proteínas do Olho/análise , Junções Intercelulares/análise , Cristalino/análise , Glicoproteínas de Membrana , Proteínas de Membrana/análise , Aquaporinas , Comunicação Celular , Membrana Celular/análise , Técnicas Imunológicas , Cristalino/imunologia , Peso MolecularRESUMO
In reassessing the role of government, many Americans have agreed that public expenditures should be curtailed. Although our universities must bear their full share of the sacrifices, some of the Administration's recent proposals would be to the detriment of the country. Drastic cuts in student aid, for example, will not encourage young people to seek the best possible education and training; reductions in federal funds for scientific instruments and facilities will mean that our laboratories will deteriorate, our accomplishments will be fewer. The success of American science has depended heavily on the talent that came from Europe at the time of World War II. Today that source has largely disappeared, and if we cannot replace it with exceptional young investigators of our own, the quality of our universities and the vitality of our science will diminish.
RESUMO
It has been demonstrated by autoradiography that radioactive amino acids serve as precursors for proteins which are subsequently incorporated into retinal rod outer segment discs in mature animals. By the isolation and purification of visual pigment from retinas of adult frogs after injection of tritiated leucine and phenylalanine, it has been shown that at least part of this labeled protein consists of visual pigment (rhodopsin).
Assuntos
Proteínas do Olho/biossíntese , Retina/metabolismo , Pigmentos da Retina/metabolismo , Animais , Anuros , Autorradiografia , Cromatografia em Gel , Leucina/metabolismo , Fenilalanina/metabolismo , Polissacarídeos , Pigmentos da Retina/análise , Células Receptoras Sensoriais/anatomia & histologia , TrítioRESUMO
The soluble crystallins in lenses from diet-restricted and control mice of diverse ages (2, 11, or 30 months) were studied by high-performance liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Results obtained with both methods suggest that dietary restriction decelerates age-related loss of soluble gamma crystallins.
Assuntos
Envelhecimento , Cristalinas/fisiologia , Dieta , Cristalino/fisiologia , Animais , Cromatografia Líquida de Alta Pressão , Cristalinas/análise , Eletroforese em Gel de Poliacrilamida , Cristalino/análise , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , RatosRESUMO
Mice homozygous for the retinal degeneration slow (rds) mutation exhibit abnormal development of photoreceptor cells, followed by their slow degeneration. We have recently cloned the rds gene and determined the structure of the wild-type rds mRNA. Here we show that the gene is expressed exclusively in photoreceptor cells. We demonstrate that it encodes a 39 kd membrane-associated glycoprotein that is restricted to photoreceptor outer segments. By electron microscopy, we show that the rds protein is distributed uniformly within outer segment discs. The developmental appearance of the rds protein coincides with outer segment disc formation. We propose that the rds protein functions as an adhesion molecule for stabilization of the outer segment discs.
Assuntos
Proteínas do Olho/genética , Proteínas de Filamentos Intermediários , Glicoproteínas de Membrana/genética , Proteínas do Tecido Nervoso , Células Fotorreceptoras/metabolismo , Degeneração Retiniana/genética , Sequência de Aminoácidos , Animais , Membrana Celular/química , DNA/genética , Proteínas do Olho/análise , Expressão Gênica , Glicosilação , Immunoblotting , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Mutantes , Microscopia Eletrônica , Dados de Sequência Molecular , Periferinas , Células Fotorreceptoras/química , Retina/química , Segmento Externo da Célula Bastonete/química , Segmento Externo da Célula Bastonete/ultraestruturaRESUMO
retinal degeneration slow (rds) is a semidominant mutation of mice with the phenotype of abnormal development of rod and cone photoreceptors, followed by their slow degeneration. The rds gene has been putatively cloned and its novel protein product initially characterized biochemically. In the present study we undertook to correct in vivo the retinal phenotype of mice with the rds mutation. We assembled a transgene containing a regulatory segment of the opsin gene positioned upstream of the wild-type rds coding region. Mice from three transgenic lines, homozygous for the rds mutation, were analyzed for expression of the transgene and for their retinal phenotypes. In two high expressing lines, we observed complete reversion to wild-type retinal morphology. In a third, low expressing line, we observed a retinal phenotype intermediate between wild type and rds/rds, suggesting partial rescue of the mutation. These results constitute formal proof that we have cloned the rds gene.
Assuntos
Proteínas do Olho/genética , Proteínas de Filamentos Intermediários , Glicoproteínas de Membrana , Camundongos Transgênicos/genética , Degeneração Neural/fisiologia , Proteínas do Tecido Nervoso , Células Fotorreceptoras/fisiologia , Displasia Retiniana/genética , Displasia Retiniana/fisiopatologia , Animais , Northern Blotting , Southern Blotting , DNA/genética , Proteínas do Olho/fisiologia , Expressão Gênica/genética , Homozigoto , Camundongos , Microscopia Eletrônica , Mutação/genética , Periferinas , Fenótipo , Células Fotorreceptoras/ultraestrutura , RNA Mensageiro/genética , Retina/patologia , Retina/fisiologia , Retina/ultraestrutura , Opsinas de BastonetesRESUMO
The regional susceptibility of the retina to diseases has been well known by clinicians for many years. It is surprising that the implications of these observations have not spawned major research efforts to characterise the structural and functional attributes of the outer retina in different regions of a foveate retina. Without such an effort, the understanding of the disease mechanisms in retinal dystrophies will remain limited and may hamper therapeutic efforts. That outer retinal disease is responsible for over 50% of blind registration in the western world underlines the importance of these considerations.
Assuntos
Macula Lutea/fisiopatologia , Doenças Retinianas/fisiopatologia , HumanosRESUMO
Clinical and preclinical studies suggest that retinoids can inhibit the growth of a small percentage of human renal cancers (RCs), although the majority of RCs both in vitro and in vivo are retinoid resistant. Our recent studies indicate that the metabolism of retinol to retinyl esters is greatly reduced in human carcinoma cell lines of the oral cavity, skin, and breast as compared with their normal epithelial counterparts, suggesting that human carcinoma cells are retinoid deficient relative to normal epithelial cells. We considered whether retinoid resistance in RCs was related to an abnormality in retinoid metabolism. The metabolism of [3H]retinol and of [3H]retinoic acid (RA) was examined in RC cell lines and normal human kidney (NK) epithelial cells cultured in media, in RA, or in RA plus IFN-alpha. The expression of LRAT (lecithin:retinol acyltransferase) was assessed by Northern and Western analysis. Retinol and retinyl ester levels were determined in tissue samples of normal human kidney and renal cell carcinoma. NK cells esterified all of the 50 nM [3H]retinol in which they were cultured. In contrast, six of the seven RC cell lines metabolized only trace amounts of [3H]retinol to [3H]retinyl esters. Consistent with this relative lack of [3H]retinol esterification by the tumor cells, the tumor cells exhibited LRAT transcripts of aberrantly low sizes relative to those in normal epithelial cells. Moreover, the NK cells expressed abundant levels of LRAT protein by Western analysis, whereas the RC cells did not express LRAT protein. When samples of human kidney tumor tissue were compared with samples of normal kidney tissue from patients who had undergone surgery for primary RC, the normal kidney tissues contained much higher levels of retinol and retinyl esters (approximately 0.5-2 microg/gram wet weight) than the tumor tissues in all seven patients examined. Culture of the RC lines in IFN-alpha plus all-trans-RA, a combination therapy used clinically, resulted in higher intracellular levels of [3H]retinol and [3H]retinyl esters. The metabolism of [3H]RA was also examined in these RC lines versus NK cells. Although the NK epithelial cells metabolized [3H]RA, the majority of the RC lines metabolized [3H]RA at a much slower rate. Most of the RC lines metabolized only 10-30% of the 50 nM [3H]RA over 6 h of culture. These data indicate that RCs both in vitro and in vivo are retinol and retinyl ester deficient relative to the normal human kidney, and they suggest that the aberrant differentiation of the neoplastic renal cells results in part from a defect in retinoid metabolism.
Assuntos
Carcinoma de Células Renais/metabolismo , Neoplasias Renais/metabolismo , Tretinoína/análogos & derivados , Vitamina A/metabolismo , Aciltransferases/biossíntese , Aciltransferases/genética , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Carcinoma de Células Renais/patologia , Divisão Celular/efeitos dos fármacos , Sistema Enzimático do Citocromo P-450/biossíntese , Sistema Enzimático do Citocromo P-450/genética , Ésteres/metabolismo , Feminino , Expressão Gênica , Humanos , Interferon alfa-2 , Interferon-alfa/farmacologia , Neoplasias Renais/patologia , Túbulos Renais Proximais/metabolismo , Masculino , Pessoa de Meia-Idade , Proteínas Recombinantes , Ácido Retinoico 4 Hidroxilase , Tretinoína/metabolismo , Tretinoína/farmacologia , Células Tumorais CultivadasRESUMO
OBJECTIVES: Although vertebroplasty is very effective for relieving acute pain from an osteoporotic vertebral compression fracture, not all patients who undergo vertebroplasty receive the same degree of benefit from the procedure. In order to identify the ideal candidate for vertebroplasty, pre-operative prognostic demographic or clinico-radiological factors need to be identified. The objective of this study was to identify the pre-operative prognostic factors related to the effect of vertebroplasty on acute pain control using a cohort of surgically and non-surgically managed patients. PATIENTS AND METHODS: Patients with single-level acute osteoporotic vertebral compression fracture at thoracolumbar junction (T10 to L2) were followed. If the patients were not satisfied with acute pain reduction after a three-week conservative treatment, vertebroplasty was recommended. Pain assessment was carried out at the time of diagnosis, as well as three, four, six, and 12 weeks after the diagnosis. The effect of vertebroplasty, compared with conservative treatment, on back pain (visual analogue score, VAS) was analysed with the use of analysis-of-covariance models that adjusted for pre-operative VAS scores. RESULTS: A total of 342 patients finished the 12-week follow-up, and 120 patients underwent vertebroplasty (35.1%). The effect of vertebroplasty over conservative treatment was significant regardless of age, body mass index, medical comorbidity, previous fracture, pain duration, bone mineral density, degree of vertebral body compression, and canal encroachment. However, the effect of vertebroplasty was not significant at all time points in patients with increased sagittal vertical axis. CONCLUSIONS: For single-level acute osteoporotic vertebral compression fractures, the effect of vertebroplasty was less favourable in patients with increased sagittal vertical axis (> 5 cm) possible due to aggravation of kyphotic stress from walking imbalance.Cite this article: Y-C. Kim, D. H. Bok, H-G. Chang, S. W. Kim, M. S. Park, J. K. Oh, J. Kim, T-H. Kim. Increased sagittal vertical axis is associated with less effective control of acute pain following vertebroplasty. Bone Joint Res 2016;5:544-551. DOI: 10.1302/2046-3758.511.BJR-2016-0135.R1.
RESUMO
The complete 3' UTR sequence encoded by the human aquaporin-1 gene is reported. The sequence encompassed by two cDNA clones showed, 33 nucleotides of 5' UTR sequence, a coding sequence of 807 nucleotides and 1886 nucleotides corresponding to the complete 3' UTR sequence. High similarity with 3' UTR sequences from rat and mouse counterparts was found. Northern blot analysis of several human tissues revealed a 2.8 kbp transcript. These data confirm the existence of water channels in the human retinal pigment epithelium.
Assuntos
Aquaporinas , Canais Iônicos/genética , Epitélio Pigmentado Ocular/química , Animais , Aquaporina 1 , Sequência de Bases , Antígenos de Grupos Sanguíneos , Northern Blotting , Química Encefálica , DNA Complementar/química , Fibroblastos/química , Humanos , Camundongos , Dados de Sequência Molecular , Ratos , Alinhamento de SequênciaRESUMO
Detergent-solubilized plasma membrane protein of either adult bovine or calf lens and high-performance liquid chromatography-purified major intrinsic protein (MIP) of the lens were reconstituted into unilamellar vesicles and planar lipid bilayers. Freeze-fracture studies showed that the density of intramembrane particles in the vesicles was proportional to the protein/lipid ratio. At high ratios, these particles crystallized into tetragonal arrays as does MIP in lens fibers. Channels induced by either purified MIP or detergent-solubilized protein had essentially identical properties. The conductance of multichannel membranes was maximal near 0 mV and decreased to 0.49 +/- 0.08 of the maximum value at voltages greater than 80 mV. The dependence of the conductance on voltage was well fit by a two-state Boltzmann distribution. Voltage steps greater than 30 mV elicited an ohmic current step followed by a slow (seconds) biexponential decrease. The amplitudes and time constants depended on the magnitude but not the sign of the voltage. Steps from 100 mV to voltages less than 30 mV caused the channels to open exponentially with a millisecond time constant. Analysis of latency to first closure after a voltage step gave nearly the same time constants as multichannel kinetics. Single-channel conductance is proportional to salt concentration from 0.1 to 1.0 M in KCl. In 0.1M KCl, the channel had two preferred conductance states with amplitudes of 380 and 160 pS, as well as three additional substates. Multi- and single-channel data suggest that the channel has two kinetically important open states. The channel is slightly anion selective. The properties of the channel do not vary appreciably from pH 7.4 to 5.8 or from pCa 7 to 2. We propose that a channel with these properties could contribute to maintenance of lens transparency and fluid balance.
Assuntos
Proteínas do Olho/metabolismo , Canais Iônicos/metabolismo , Glicoproteínas de Membrana , Animais , Aquaporinas , Bovinos , Cromatografia Líquida de Alta Pressão , Condutividade Elétrica , Proteínas do Olho/química , Proteínas do Olho/isolamento & purificação , Técnica de Fratura por Congelamento , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Canais Iônicos/química , Cinética , Estrutura Molecular , Cloreto de PotássioRESUMO
Cellular retinaldehyde-binding protein (CRALBP) is abundant in the retinal pigment epithelium (RPE) and Müller cells of the retina where it is thought to function in retinoid metabolism and visual pigment regeneration. The protein carries 11-cis-retinal and/or 11-cis-retinol as endogenous ligands in the RPE and retina and mutations in human CRALBP that destroy retinoid binding functionality have been linked to autosomal recessive retinitis pigmentosa. CRALBP is also present in brain without endogenous retinoids, suggesting other ligands and physiological roles exist for the protein. Human recombinant cellular retinaldehyde-binding protein (rCRALBP) has been over expressed as non-fusion and fusion proteins in Escherichia coli from pET3a and pET19b vectors, respectively. The recombinant proteins typically constitute 15-20% of the soluble bacterial lysate protein and after purification, yield about 3-8 mg per liter of bacterial culture. Liquid chromatography electrospray mass spectrometry, amino acid analysis, and Edman degradation were used to demonstrate that rCRALBP exhibits the correct primary structure and mass. Circular dichroism, retinoid HPLC, UV-visible absorption spectroscopy, and solution state 19F-NMR were used to characterize the secondary structure and retinoid binding properties of rCRALBP. Human rCRALBP appears virtually identical to bovine retinal CRALBP in terms of secondary structure, thermal stability, and stereoselective retinoid-binding properties. Ligand-dependent conformational changes appear to influence a newly detected difference in the bathochromic shift exhibited by bovine and human CRALBP when complexed with 9-cis-retinal. These recombinant preparations provide valid models for human CRALBP structure-function studies.
Assuntos
Proteínas de Transporte/química , Sequência de Aminoácidos , Animais , Bovinos , Cromatografia Líquida de Alta Pressão , Dicroísmo Circular , Temperatura Alta , Humanos , Luz , Espectrometria de Massas , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Fragmentos de Peptídeos/análise , Ligação Proteica , Desnaturação Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes , Retinoides/metabolismo , Espectrofotometria Ultravioleta , Relação Estrutura-AtividadeRESUMO
We have characterized cDNA clones encoding the alpha 1 and beta 1 subunits of Na,K-ATPase produced in the human retinal pigment epithelium (hRPE). In addition to isolating clones corresponding to known sequences of Na,K-ATPase subunits, we report hitherto unknown forms of Na,K-ATPase with unique deduced amino acid (aa) sequences in their C-termini. Truncated cDNA sequences were found for both the beta 1 and alpha 1 subunits. While the beta 1 sequence is truncated by two aa residues at the C terminus, in the alpha 1 sequence 342 aa have been replaced by a unique sequence containing only 44 aa. Interestingly, this new C-terminal polypeptide shows sequence similarities to the Ca(2+)-ATPase and contains consensus sequence elements for phosphorylation and cell adhesion, suggesting expression of Na,K-ATPase subunits with unique functions. Using reverse transcription-polymerase chain reaction, RNA sequences for alpha 1, beta 1 and their corresponding truncated isoforms were quantified. 4.0 x 10(5) alpha 1 and 2.3 x 10(5) beta 1 molecules were found per ng of mRNA from hRPE. Much lower levels were detected for truncated alpha 1 and beta 1 (3.6 x 10(3) and 2.7 x 10(3) molecules/ng, respectively). These data corroborate the expression of truncated transcripts coding for unique aa sequences in hRPE, and suggest that factors other than alpha 1 and beta 1 mRNA levels regulate the equimolar accumulation of alpha and beta subunits in the plasma membrane.
Assuntos
RNA/genética , ATPase Trocadora de Sódio-Potássio/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Humanos , Dados de Sequência Molecular , Epitélio Pigmentado Ocular/enzimologia , RNA/análise , Alinhamento de Sequência , Análise de Sequência , ATPase Trocadora de Sódio-Potássio/biossínteseRESUMO
Na,K-ATPase in the retinal pigment epithelium (RPE) is apically localized, whereas in most other tissues this pump is found predominantly in the basolateral membrane domain. As part of our investigations into the molecular aspects of this pump in the RPE, we have cloned the cDNA and characterized the expression of the gene encoding the beta 2 subunit isoform of Na,K-ATPase in human, rat and bovine RPE and in the bovine choroid plexus. We have also detected the beta 2 isoform polypeptide in the human RPE (hRPE). Comparison of complete coding sequences derived from cloned cDNAs revealed that all beta 2 sequences from RPE, and the choroid plexus, differed uniformly at positions: P51/L, M121/I, and L148/R from the published sequences for human retina and liver. However, analysis of 10 RT-PCR clones derived from 5 fetal and 2 adult human retinas sequenced in our laboratory, revealed that only the P51/L residue was different with the hRPE beta 2 subunit sequence. Northern blot analysis indicated a 3.4-kb RNA transcript for the beta 2 subunit, a 4.5-kb RNA for the alpha 1 subunit and a doublet of 2.3 and 2.6 kb for the beta 1 subunit, respectively. alpha 1 (100 kDa), beta 1 (45 kDa) and beta 2 (65 kDa) isoforms were detected in hRPE extracts by immunoblotting. No alpha 2 and alpha 3 RNA transcripts were found in the hRPE. Quantification of beta 2 mRNA by RT-PCR revealed 2.7 x 10(5) molecules per ng of poly A+ RNA. This is similar to the beta 1 isoform levels reported previously from our laboratory. These data demonstrate the coexistence of significant amounts of alpha 1, beta 1 and beta 2 Na,K-ATPase subunits in the RPE. It is therefore reasonable to suggest that both alpha 1 beta 1 and alpha 1 beta 2 heterodimers are present in these cells.